| ObjectiveCyr61, a member of CCN protein family, is an immediate early gene and an important regulator of cell matrixstromal. Cyr61 has obvious mitogenic and chemotactic activity, involved in the regulation of cell proliferation, transfer, adhesion and the formation of blood vessels. Cyr61 express in a variety of biological functions in tumor, and Cyr61 expression levels and functions are different in different tumor tissues and cells.However, the exact relationship between Cyr61 and the tumor and its mechanism is not clear. In this study, we analysed Cyr61 expression in Human Hepatocellular Carcinoma (HCC), neighboring non-cancerous tissues, hepatic cirrhosis tissues and normal adult livers. Then we analyzed the expression and relations between Cyr61 and VEGF in human hepatocellular carcinoma and hepatic cirrhosis tissues and their contribution to development of HCC. At last we observed the proliferation and migration of human hapetocelluar carcinoma cell line HepG2 after the introduction of exogenous Cyr61. This study established the foundation for further studies on the function and mechanism of Cyr61 in tumorigenesis, and provided a new tumor marker and a gene therapy target for tumor diagnosis and therapy.Methods1. The expression of Cyr61 protein was determined by immunohistochemical staining (SP) in 5 samples of health people and 35 samples of human HCC. The relationship between these expression levels and clinical pathological features was evaluated.2. The expression of Cyr61 of liver tissues of health people, human HCC and neighboring non-cancerous were quantitatively analyzed by Western blot.3. The expression of Cyr61 level of mRNA were determined by RT-PCR in 30 samples of human HCC and neighboring non-cancerous tissues, then analyzed their differences.4. Expression of Cyr61 and VEGF in 58 cases of HCC and hepatic cirrhosis tissues was observed by immunohistochemistry SP. The Cyr61 and VEGF expression of protein were analyzed and compared with clinical pathologic characters in 28 samples of human HCC. The relativity between Cyr61 and VEGF was also analyzed in the process of HCC formation.5. Recombinant Ad-Cyr61 and Ad-RFP were amplified by 293 cells, then take titer determination and cell infection. Then we got transient high expressed cell model of Ad-Cyr61 HepG2 and Ad-RFP HepG2.6. The protein and mRNA expression of Cyr61 in Cyr61-HepG2cells,RFP -HepG2 cells and HepG2 cells were observed by immunohistochemistry, Western blot and RT-PCR.7. MTT, cell counting, plate colony formation and cell wound healing assay were performed to determine the Cyr61's effect on the proliferation and migration of HepG2.Results1. The expression of Cyr61 protein was significantly higher in well-differentiated HCC than in poorly differentiated HCC (P < 0.01); The expression of Cyr61 was higher in HCC tissue with vein infiltrating and multinodular than in that with no vein infiltrating and mononodular ( P < 0.05).2. The expression of Cyr61 protein was negative in liver tissues of health people, and the expression of Cyr61 protein was higher in tissues neighboring non-cancerous than tissues of human HCC.3. Cyr61 mRNA was positive in all 30 samples of human HCC. The expression of Cyr61 mRNA was lower in tumor tissues than in neighboring non-cancerous tissues (P < 0.05). The expression of Cyr61 mRNA in health liver tissues is no differences with that in HCC,and lower than that in tissues neighboring non-cancerous(P<0.05).4. Cyr61 and VEGF expressed higher in hepatic cirrhosis than HCC tissues (P<0.01). Expression of Cyr61 and VEGF was relative to metaptosis of HCC (P<0.05). Cyr61 and VEGF have relativity in HCC and hepatic cirrhosis tissues by correlation test(r=0.444, P<0.01).5. Cell line HepG2 infected by Ad-Cyr61 increased in the number of cells and colony-forming, and cells scratches healed gradually compared with control group.Conclusion1. The expression of Cyr61 protein and level of mRNA and the relationship between these expression levels and clinical pathological features suggested that Cyr61 may play roles significantly in earlier period of human HCC and may correlate with matrix reconstititution, invasion and metastasis of human HCC.2. Expression of Cyr61 was related with expression of VEGF. Abnormal expression of Cyr61 and VEGF are relative with the development of HCC and have significant contribution in angiogenesis of HCC and transforming hepatic cirrhosis to HCC, which may provide a relatively objective indicator to early diagnosis of HCC.3. Exogenous Cyr61 stimulated the proliferation and migration of HCC cells, which suggested Cyr61 may play a role in tumorigenesis and metastasis. |
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